PROJECT SUMMARY/ABSTRACT Chronic gastric inflammation, typically caused by Helicobacter pylori (H. pylori), is the most consistent lesion leading to cancer. During a well-choreographed interaction between H. pylori and the host, the progression from chronic inflammation to cancer involves gastric epithelial changes with evidence of hyperproliferation and the disruption of normal gland morphology and differentiation. Although H. pylori virulence factors are known to interfere with signaling pathways in gastric epithelial cells, the identity of these target cells is unknown. Abnormal differentiation (metaplasia) is associated with cancer and seems to reflect a permanent alteration in the behavior of the stem cells, thus making the gastric stem cell a candidate H. pylori target. The objective of this proposal is to identify the underlying mechanism by which H. pylori-host interactions trigger the disruption of epithelial cell differentiation and thus the cascade leading to cancer. The acquisition of such knowledge is the first step in a continuum of research required to achieve our long-term goal that is to understand the pathogenesis of H. pylori-induced gastric cancer. The central hypothesis is that H. pylori primarily triggers hyperproliferation of the stem cell compartment in both the fundus and antrum leading to aberrant epithelial cell differentiation. Moreover we hypothesize that differences in fundic pathology triggered by H pylori are due to differences in target cell activation in the fundus versus antrum. The hypothesis has been formulated on the basis of preliminary studies produced in our laboratory demonstrating the first successful efforts to generate three-dimensional human gastric organoids (hGOs) de novo through directed differentiation of human pluripotent stem cells (hPSCs). hGOs are antral in nature and can be used to effectively model the early stages of H. pylori infection. The rationale for these studies is to acquire an understanding of the molecular mechanisms by which H. pylori infection colonizes the stomach and induces disease. Acquiring such knowledge may allow us to then develop techniques to disrupt bacterial colonization and thus prevent disease progression. Guided by strong preliminary data, this hypothesis is tested by pursuing two specific aims: 1) What are the primary H. pylori target cells during infection of the antrum? and 2) What are the molecular pathways that regulate development of fundic organoids? The hypothesis will be tested using genetic lineage tracing and single-cell RNA-sequencing. The research proposed is innovative, because it focuses on novel approaches that will allow us to assay changes in gastric epithelial cell proliferation and differentiation in relation to the direct interaction with H. pylori. Conceptually innovative, the current proposal will identify novel insights into the direct interaction between H. pylori and the host gastric epithelium independent of the immune response. Moreover the current study has the potential to advance our understanding of the mechanism by which H. pylori may alter the gastric stem cell fate. The proposed research is significant because it is expected to provide knowledge that is crucial to our understanding of the role of H. pylori as a carcinogen.